Protein preparations

ABSTRACT

A plastein material is made by reversing the normal hydrolytic activity of a serine protease. The protease produces a plastein material by acting on a proteinaceous substrate. The substrate is preferably whey, casein or soy protein.

FIELD OF INVENTION

The present invention relates to a method for increasing the viscosityof a reaction mixture, plastein materials obtained by this method, andfood products comprising these plastein materials.

BACKGROUND OF THE INVENTION

Non-bitter protein preparations are obtainable by performing a reversedhydrolysis. Proteolytic enzymic reactions are reversible as are otherenzymic reactions. A reversal of the enzymic degradation of peptidebonds is termed a plastein reaction, and a protein-like productsynthesized by a plastein reaction is termed plastein or plasteinmaterial [vide e.g. Horowitz, J. & Haurowitz, F. (1959); Biochim.Biophys. Acta., 33, 231-237; and Determann et al. (1963); Helv. Chim.Acta., 46, 2498]. Plastein material is different from protein and may beregarded as a mixture of high molecular polypeptides. In fact Wieland etal. [Wieland, T; Determann, H. & s Albrecht, E. (1960); Ann., 633, 185 ]define plastein reaction as the formation of high molecularpolypeptides.

Various microbial proteases are known to posses plastein syntheticactivities. Well known proteases also known for their plastein syntheticactivities are pepsin, α-chymotrypsin, trypsin, and papain [vide e.g.Fujimaki, M.; Kato, H.; Arai, S. & Yamashita, M. (1971); J. Appl. Bact.34(1), 119-131].

At least four points are hitherto believed to be complied with for theplastein reaction to proceed effectively. First, the concentration ofsubstrate should be high. Second, the substrate should be of lowmolecular weight. Third, the pH for synthesis of the plastein isdifferent than for hydrolysis of the protein. The pH range for synthesisof plastein is narrower than the pH range for hydrolysis. Fourth, arelatively long incubation time should be applied with.

Apart from removing bitterness of hydrolysates, the plastein reactionhas other food processing potentials, e.g. preparing gel-like productswith excellent visco-elastic properties for incorporation in differenttypes of foods, preparing products with improved amino acid compositionusing mixtures of hydrolysates as substrates, preparing products withvery high level of a single amino acid which could be used as a dietarysupplement to certain foods, and preparing special types of solublepeptides having important flavour or other characteristics.

SUMMARY OF THE INVENTION

The present invention provides a method for increasing the viscosity ofthe reaction mixture, in which an enzyme preparation comprising aproteolytic enzyme having the following characteristics:

(a) it is a serine protease specific for glutamic acid (Glu) andaspartic acid (Asp) residues;

(b) it has a specific activity of at least 25 CPU (as defined herein)per gram of enzyme protein;

(c) it has an apparent molecular weight of about 23,600;

(d) it is inhibited by diisopropyl phosphofluoridate, but not byphenylmethane sulfonylfluoride;

(e) it exhibits 75% or more of its maximum activity in the pH range of6.5-10.0;

and which enzyme preparation is substantially free from otherproteolytic activity, is added to a proteinaceous substrate allowing aplastein reaction, followed by incubation at conditions at whichincreased viscosity occurs, and subsequent inactivation of the enzyme.

In its second aspect the invention provides plastein materials obtainedby adding to a proteinaceous material an enzyme preparation comprising aproteolytic enzyme having the following characteristics:

(a) it is a serine protease specific for glutamic acid (Glu) andaspartic acid (Asp) residues;

(b) it has a specific activity of at least 25 CPU (as defined herein)per gram of enzyme protein;

(c) it has an apparent molecular weight of about 23,600;

(d) it is inhibited by diisopropyl phosphofluoridate, but not byphenylmethane sulfonylfluoride;

(e) it exhibits 75% or more of its maximum activity in the pH range of6.5-10.0;

and which enzyme preparation is substantially free from otherproteolytic activity, followed by incubation and subsequent inactivationof the enzyme.

In its third aspect the invention provides food products comprisingplastein material of the invention.

The proteolytic enzyme defined above has previously been characterizedin U.S. Pat. No. 4,266,031 as a contaminant of subtilisin A produced byBacillus licheniformis. In this specification, however, the enzyme waswrongly characterised as a non-serine protease. Later investigations onthis subject have now revealed that the enzyme is in fact inhibited bydiisopropyl phosphofluoridate (DFP), and is therefore a serine protease.

Furthermore, there is no indication of the specific proteolytic activityof the enzyme in the above-mentioned US patent, and its utility for usein a process of the invention for increasing the viscosity of thereaction mixture is therefore not anticipated by the disclosure of theenzyme per se in this patent.

In International Patent Application No. WO 91/13554 it has been foundthat the proteolytic enzyme defined above provides for limited andspecific hydrolysis of proteins at Glu and/or Asp residues, and theapplication, therefore, relates to the use of the enzyme in a method forhydrolysis of proteins.

Now it has surprisingly been found that the proteolytic enzyme definedabove possesses excellent plastein synthetic activities, particularlywhen applied to whey protein, which enables this protease to exertincreased viscosity of the reaction mixture, even at moderateconditions.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to theaccompanying drawings, in which:

FIG. 1 is a graph showing the activity (% rel.) in relation to pH (pH6-11) of the SP 446 protease of the invention;

FIG. 2 is a graph showing the activity (% rel.) in relation totemperature (15°-70° C.) of the SP 446 protease of the invention in thepresence (white squares) and absence (black squares) of sodiumtripolyphosphate (STPP);

FIG. 3 shows the cleavage of insulin by the SP 446 protease;

FIG. 4 shows the viscosity and Δ osmolality during incubation of wheyprotein isolate with SP 446 protease of the invention (□=mPa*s;=ΔmOsm/kg);

FIG. 5 shows the viscosity (mPa*s) in relation to degree of hydrolysis(% DH), during incubation of whey protein isolate with SP 446 proteaseof the invention;

FIG. 6 shows the viscosity (mPa*s) during incubation of soy protein withSP 446;

FIG. 7 shows the molecular weight distribution of plastein material (6%DH) obtained according to Ex. 2 (--Peptide distribution;--Accumulatedweight distribution); and

FIG. 8 shows the distribution of peptides of plastein material (6% DH)obtained according to Ex. 2 ( Distribution by weight; □ Distribution bynumber).

DETAILED DISCLOSURE OF THE INVENTION

The present invention relates to the use of specific proteolytic enzymesfor plastein synthesis, i.e. a process for increasing the viscosity ofthe reaction mixture.

Accordingly, the invention provides a method for increasing theviscosity of the reaction mixture, in which an enzyme preparationcomprising a proteolytic enzyme having the following characteristics:

(a) it is a serine protease specific for glutamic acid (Glu) andaspartic acid (Asp) residues;

(b) it has a specific activity of at least 25 CPU (as defined herein)per gram of enzyme protein;

(c) it has an apparent molecular weight of about 23,600;

(d) it is inhibited by diisopropyl phosphofluoridate, but not byphenylmethane sulfonylfluoride;

(e) it exhibits 75% or more of its maximum activity in the pH range of6.5-10.0;

which enzyme preparation is substantially free from other proteolyticactivity, is added to a proteinaceous substrate allowing a plasteinreaction, followed by incubation at conditions which cause increasedviscosity, and subsequent inactivation of the enzyme.

The Enzyme

The proteolytic enzyme employed in the present method may be oneproducable by a microorganism, in particular a bacterium. Such abacterium may be a strain of Bacillus licheniformis, e.g. a strain knownto produce subtilisin A as well as another protease corresponding to theproteolytic enzyme defined above. In this case, the proteolytic enzymemay be prepared by culturing the bacterial strain under conditionsconducive to the production of alkaline protease which may then beisolated, after which the protease activities may be separated bymethods known per se, e.g. by the process described in theabove-mentioned U.S. Pat. No. 4,266,031.

The strain of Bacillus licheniformis may also be a mutant strain, suchas a mutant in which the gene encoding subtilisin A has beeninactivated, for instance by conventional mutagenesis proceduresinvolving the use of a mutagen such as nitrosoguanidine, e.g.substantially by the procedure disclosed in the abovementioned U.S. Pat.No. 4,266,031 (disclosing the inactivation of the gene encoding theproteolytic enzyme of current interest). Alternatively, the inactivationof the subtilisin A gene may also take place by recombinant DNAtechniques, e.g. by inserting one or more nucleotides into thesubtilisin A gene so as to disrupt the sequence. This may for instancebe done by homologous recombination, e.g. as described by Ferrari etal., J. Bacteriol. 154(3), 1983, pp. 1513-1515. The proteolytic enzymemay also be produced by isolating the DNA sequence from a cDNA orgenomic library of microorganism producing the enzyme, e.g. a strain ofBacillus licheniformis, inserting the DNA sequence into a suitableexpression vector, transforming a suitable host microorganism with thevector, growing the host under conditions which are conducive to theproduction of the enzyme and recovering the enzyme from the culture.These steps may be carried out by standard procedures, cf. Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982.

In a particular embodiment of the present process, the proteolyticenzyme is one which has the amino acid sequence ID No. 1 shown in theappended sequence listing, or a derivative thereof.

In the present context, the term "derivative" is understood to indicatea proteolytic enzyme which is derived from the native enzyme by additionof one or more amino acids to either or both the C- and N-terminal endof the native protein, substitution of one or more amino acids at one ora number of different sites in the native amino acid sequence, deletionof one or more amino acids at either or both ends of the native proteinor at one or more sites in the amino acid sequence, or insertion of oneor more amino acids at one or more sites in the native amino acidsequence, provided that the proteolytic activity of the enzyme is notthereby impaired.

In the context of this invention, proteolytic activity is expressed interms of casein protease units (CPU), cf. Example 1 for definition.

The enzyme may be added to the proteinaceous material in a concentrationin the range 0.05-50 CPU/100 g protein, more preferably 0.1-25 CPU/100 gprotein, most preferably 1-25 CPU/100 g protein.

The Substrate

The proteinaceous material, which may advantageously be subjected to amethod of the invention, may be any of the proteins or proteinaceousmaterials suggested for hydrolysis in the prior literature. Examples ofsuitable proteinaceous materials are animal proteins such as wheyprotein, casein, meat proteins, fish protein, red blood cells, egg whiteor gelatin, or vegetable proteins such as soy protein, grain proteins,e.g. wheat gluten or zein, rape seed protein, alfalfa protein, peaprotein, fabaceous bean protein, cotton seed protein or sesame seedprotein, or a combination hereof.

In a specific embodiment, the proteinaceous material is casein, soyprotein, or whey protein, or a proteinaceous mixture comprising one ormore of these proteins.

Preferably the proteinaceous material is whey protein, or aproteinaceous mixture comprising whey protein.

During incubation, the substrate concentration may be within the range5-50% (w/w), more preferably 5-30% (w/w), most preferably 5-15% (w/w).

Incubation Conditions

During incubation, reaction conditions that make allowance for optimumplastein synthetic activities should be observed. In contrast to otherplastein reactions known, the method of the invention may be performedat moderate conditions.

The method may be performed at an incubation temperature within therange 20°-70° C., preferably 45°-65° C.

The incubation may be performed at a pH within the range 4-12, morepreferred 6-9, most preferred around pH 8.

The incubation time may be in the range 0.5-24 hours, more preferred1-10 hours, most preferred 1-5 hours.

For preparation of products with improved amino acid composition, orproducts with very high level of a single amino acid, the substrate maybe a mixture of hydrolysates, or the substrate may be added one or moresingle amino acid(s).

In accordance with established practice, the proteolytic enzyme maysuitably be inactivated by increasing the temperature of the incubationmixture to above about 70° C., preferably 85° C. for approximately 3minutes, or by decreasing the pH of the incubation mixture to belowabout 5.0.

Plastein Materials

In another aspect, the invention relates to protein preparationsobtained by the method of invention. Accordingly, the invention providesplastein materials obtained by adding to a proteinaceous material anenzyme preparation comprising a proteolytic enzyme having the followingcharacteristics:

(a) it is a serine protease specific for glutamic acid (Glu) andaspartic acid (Asp) residues;

(b) it has a specific activity of at least 25 CPU (as defined herein)per gram of enzyme protein;

(c) it has an apparent molecular weight of about 23,600;

(d) it is inhibited by diisopropyl phosphofluoridate, but not byphenylmethane sulfonylfluoride;

(e) it exhibits 75% or more of its maximum activity in the pH range of6.5-10.0;

which enzyme preparation is substantially free from other proteolyticactivity, followed by incubation and subsequent inactivation of theenzyme.

In a more specific aspect, the plastein material is obtained fromcasein, soy protein, or whey protein, or a proteinaceous mixturecomprising one or more of these proteins.

Preferably, the plastein material is obtained from whey protein, orproteinaceous mixtures comprising whey protein.

In another specific aspect, the plastein material has a molecular weightnot exceeding approximately 7,000.

In a further specific aspect, the plastein material has a dry mattercomposition essentially similar to that of the starting material (thesubstrate).

In a yet further aspect, the plastein material has a viscosity at 50° C.of more than 40 mPa*s, preferably more than 60 mPa*s, most preferredmore than 80 mPa*s, as determined by a HAAKE VT 181 viscosimeter with NVsensor and spindle No. 4.

For preparation of plastein material with improved amino acidcomposition, or plastein material with very high level of a single aminoacid, the substrate may be a mixture of hydrolysates, or the substratemay be added one or more of the desired single amino acid(s). Byenriching the amino acid composition food products of increased nutrientvalue may be obtained.

The plastein material of the invention possesses excellent emulsifyingcapacity. Generally, increased viscosity leads to more stable emulsions.However, increased viscosity not always leads to improved emulsifyingcapacity, as is the case with plastein material of this invention.

High molecular compounds are associated with an increased risk ofallergenicity. Therefore, an increased degree of hydrolysis generallyleads to less allergenic products. As evident from FIG. 7, the plasteinmaterial described has a very specific molecular weight distribution.The plastein material does not contain peptides having a molecularweight exceeding approximately 7,000. Hence, the plastein materialpossesses reduced risk of allergenicity, which allows for implementationinto particular mother milk substitutes.

Moreover, the plastein material of the invention is neutral in respectto taste, which also allows for implementation into various foodproducts, e.g. emulsified meat products, low fat spreads, or mother milksubstitutes.

Food Products

in a further aspect, the invention relates to food products comprisingplastein material obtained by the method of the invention.

Advantageously, the plastein material of the invention may beimplemented in the manufacture of e.g. gel-like products with specialvisco-elastic properties for incorporation in different types of foods,such as emulsified meat products, low fat spreads, whipped toppings anddesserts, products with especially designed nutritive values, e.g. dueto improved amino acid compositions or increased levels of single aminoacids, which could be used as a dietary supplement to certain foods,products containing special types of soluble peptides having importantflavour or other characteristics, etc.

In a preferred embodiment, the plastein material of the invention may beincorporated into mother milk substitute,.

The amount of plastein material incorporated in the food product willtypically be in the range of 1-30% (w/w). The food product may comprisefat and/or carbohydrates, and may further comprise usual food additives,such as flavouring agents, sweeteners, vitamins, minerals and traceelements.

The invention is further illustrated in the following examples, whichare not intended to be in any way limiting to the scope of the inventionas claimed.

Characterization of Bacillus licheniformis SP 446 protease Yield of SP446 protease

Alcalase™ PPA 1618 was purified as described in U.S. Pat. No. 4,266,031.The yield of purified SP 446 protease was determined by measuring theenzymatic activity of the starting and purified SP 446 protease usingCBZ-Phe-Leu-Glu-pNA (Boehringer Mannheim) as substrate. It was necessaryto add phenylmethane sulfonylfluoride (1:10 vol) in order to inactivatesubtilisin A present in the enzyme preparation, as subtilisin A is ableto degrade the substrate, apparently by cleaving after Phe or Leu. Theenzymatic activity of the starting material (40 ml) was measured in aPerkin-Elmer Lambda reader as the absorbance at 405 nm/min./ml and wasdetermined to be 166,920. The enzymatic activity of the purifiedmaterial (31 ml) was similarly measured and determined to be 158,720.Thus, the yield of SP 446 protease was 95%.

Proteolytic activity

The proteolytic activity of the SP 446 protease was determined to be 27CPU/g using casein as substrate. 1 casein protease unit (CPU) is definedas the amount of enzyme liberating 1 millimole of primary amino groups(determined by comparison with a serine standard) per minute understandard conditions as described below:

A 2% (w/v) solution of casein (Hammarsten®, supplied by Merck AG,Darmstadt, FRG) is prepared with the Universal Buffer described byBritton and Robinson, J. Chem. Soc., 1931, p. 1451), adjusted to a pH of9.5. 2 ml of the substrate solution are pre-incubated in a water bathfor 10 min. at 25° C. 1 ml of an enzyme solution containing b g/ml ofthe enzyme preparation, corresponding to about 0.2-0.3 CPU/ml of theUniversal Buffer (pH 9.5) is added. After 30 min. of incubation at 25°C., the reaction is terminated by the addition of a quenching agent (5ml of a solution containing 17.9 g of trichloroacetic acid, 29.9 g ofsodium acetate and 19.8 g of acetic acid made up to 500 ml withdeionized water). A blank is prepared in the same way as the testsolution with the exception that the quenching agent is added prior tothe enzyme solution. The reaction mixtures are kept for 20 min. in awater bath after which they are filtered through Whatman 42 paperfilters. A folder AF 228/1 describing this analytical method isavailable upon request from Novo Nordisk A/S, Denmark.

Primary amino groups are determined by their colour development witho-phthaldialdehyde (OPA), as follows:

7.62 g of disodium tetraborate decahydrate and 2.0 g of sodiumdodecylsulfate are dissolved in 150 ml of water. 160 mg of OPA dissolvedin 4 ml of methanol were then added together with 400 μl ofβ-mercaptoethanol after which the solution is made up to 200 ml withwater. To 3 ml of the OPA reagent are added 400 μl of the flitratesobtained above, with mixing. The optical density (OD) at 340 nm ismeasured after about 5 min. The OPA test is also performed with a serinestandard containing 10 mg of serine in 100 ml of Universal Buffer (pH9.5). The buffer alone is used as a blank. The protease activity iscalculated from the OD measurements by means of the following formula:##EQU1## wherein OD_(t), OD_(b), OD_(ser), and OD_(B) are the opticaldensities of the test solution, blank, serine standard, and buffer,respectively, C_(ser) is the concentration of serine (mg/ml) in thestandard (in this case 0.1 mg/ml), and MW_(ser) is the molecular weightof serine (105.09). Q is the dilution factor for the enzyme solution (inthis case 8) and t_(i), is the incubation time in minutes (in this case30 minutes).

pH Activity

The pH dependence of the activity of the SP 446 protease was determinedby the OPA casein method described above with the modification that theUniversal Buffer was adjusted to different pH values, i.e. pH 6, 7, 8,9, 10 and 11. The results are shown in FIG. 1 from which it appears thatthe SP 446 protease has a pH optimum in the range of pH 8-10.

Temperature activity

The temperature dependence of the activity of the SP 446 protease wasdetermined by the OPA casein method described above with themodifications that the enzyme reaction was carried out at differenttemperatures, i.e. 15° C., 30° C., 40° C., 50° C., 60° C. and 70° C.,and that the enzyme reaction was conducted in the presence and absenceof 0.1% sodium tripolyphosphate (STPP) which is a common ingredient inmany commercial detergents. The results are shown in FIG. 2 from whichit appears that the SP 446 protease has a temperature optimum of about50° C. whether STPP is present or not.

Glu specificity

The Glu specificity of the SP 446 protease was determined as follows:

0.5 ml of 1 mg/ml human insulin in Universal Buffer, pH 9.5 (videsupra), and 75 μl 446 protease (0.6 CPU/I) in the same buffer wereincubated for 120 min. at 37° C. The reaction was terminated by adding50 μl 1N hydrochloric acid.

The insulin molecule was cleaved into a number of peptide fragments.These were separated and isolated by reverse phase HPLC using a suitableC-18 column (Hibar LiChrosorb RP-18,5 μm particles provided by Merck AG,Darmstadt, FRG). The fragments were eluted with the following solvents:

A. 0.2M sodium sulfate and 0.1M phosphoric acid; pH 2.5;

B. Acetonitrile/water, 50%; on a linear gradient of from 90% A/10% B to80% A/20% B for 0-5 min. and subsequently for 50 min. with 80% A/20% B.The isolated fragments were subjected to amino acid sequencing byautomated Edman degradation, using an Applied Biosystems (Foster City,Calif., USA) Model 470A gas-phase sequencer, and the phenylthiohydantoin(PTH-) amino acids were analyzed by high performance liquidchromatography as described by Thim et al., "Secretion of human insulinby a transformed yeast cell", FEBS Letters 212(2), 1987, p.307. Thecleavage sites in the insulin molecule were identified as shown in FIG.3.

N-terminal amino acid sequence

The N-terminal amino acid sequence of the purified SP 446 protease wasdetermined as described in the foregoing section. The N-terminalsequence was determined to be SEQ ID No: 2.

Complete amino acid sequence

The complete amino acid sequence was determined from the DNA sequence.The DNA sequence was determined by standard techniques as described inthe section entitled "Detailed Disclosure Of The Invention". Thecomplete amino acid sequence is SEQ ID No: 1 shown in the appendedsequence listing.

Based on this amino acid sequence, the molecular weight of the SP 446protease was determined to be 23,600.

Inactivation of the SP 446 protease with DFP

Incubation of the enzyme with PMSF (1% in isopropanol) in a ratio of 1to 10 (by volume) did not result in any inactivation of the SP 446protease. However, incubation of 10 μl (1 mg/ml) of the enzyme with 80μl 10 mM MOPS, pH 7.2,+10 μl 0.1M diisopropyl phosphofluoridate (DFP)for 60 min. resulted in complete inactivation of the enzyme as measuredby its activity on the substrate CBZ-Phe-Leu-Glu-pNA.

EXAMPLE 2

Preparation of high viscosity whey protein

The increase in viscosity was investigated in relation to the degree ofhydrolysis (DH).

As substrate spray-dried whey protein concentrate (Lacprodan-80,available from Danmark Protein A/S, Nr. Vium, 6920 Videbaek, Denmark)was used. The protein concentration was 8.0% (w/w), and the SP 446protease dosage was 5 CPU/100 g protein. The incubation was performed at65° C. in a pH stat at pH 8.0, adjusted with NaOH.

The viscosity was monitored at 50° C. using a HAAKE VT 181 viscosimeterwith NV sensor and spindle No. 4.

The result of this test is presented in FIG. 4. It appears from thisfigure that maximum viscosity was obtained after 3 hours of incubation.Interestingly, at this point also a local minimum in osmolality appears.

From the base consumption the degree of hydrolysis was calculatedaccording to Adler-Nissen; Enzymic Hydrolysis of Food Proteins; ElsvierApplied Science Publishers Ltd. (1961), p. 122.

The degree of hydrolysis may be calculated by means of the followingformula: ##EQU2##

The total number of peptide bonds in a protein may be calculated fromits amino acid composition. The number of peptide bonds cleaved may bedetermined from an assay of the free O-amino groups in the hydrolysateby the following method using trinitrobenzene sulphonic acid (TNBS):

0.25 ml of a sample containing between 0.25×10⁻³ and 2.5×10⁻³ aminoequivalents/l is mixed in a test tube with 2.00 ml phosphate buffer atpH 8.2. 2 ml of a 0.1% TNBS solution is added and the test tube isshaken and placed in a water bath at 50°±1° C. for 60 min. Duringincubation, the test tube and water bath are covered with aluminium foilbecause the blank reaction is accelerated by exposure to light. After 60min., 4.00 ml HCl are added to terminate the reaction, and the test tubeis allowed to stand at room temperature for 30 min. before reading theabsorbance spectrophotometrically against water at 340 nm. For furtherdetails, see Adler-Nissen, J. Agric. Food Chem. 27, 1979, p. 1256-1262.

FIG. 5 presents the viscosity in relation to DH. Clearly, the productshows maximum viscosity at a DH of approximately 6%. The taste of thisproduct was evaluated, and it was found to be excellent, non-bitter,with possibilities for implementation into food or nutrient products.

EXAMPLE 3

Emulsifying Properties

The emulsifying capacity of the high viscosity product of the inventionwas examined.

Samples of the high viscosity product obtained according to Ex. 2,having a DH of 2%, 4% and 6%, respectively. The incubation wasterminated by heat treatment at 85° C. for 3 minutes at the desired DH.The samples were subjected to analysis and compared to the raw materialby two different test methods, a Swift titration method as described bySwift et al.; Food Technology (1961), 15, p.468-473, and a model foodsystem similar to a mother milk substitute.

The Swift titration was used with slight modifications.

1.25 g of protein obtained according to Ex. 2 (N×6.38) were dispersed atlow speed for two minutes using 250 ml of 0.5M NaCl. 50 ml weretransferred to a blender jar and weighed. 50 ml of soy bean oil wereadded and the oil-water mixture was subjected to high-speed cutting andmixing (approximately 13,000 rpm) with a MSE Homogenizer.

The blender jar was cooled in an ice bath. During this mixing a steadyflow of oil was added from a separating funnel at a rate of 0.3 ml persecond (The rate of oil addition has an effect on the emulsifyingcapacity). Oil was added until the emulsion formed resisted mixing. Thenthe position of the blades of the homogenizer was changed in order tosecure the absorption of fresh oil. This thickening indicates that the"end point" is about to be reached.

Addition of oil was immediately terminated when a sudden decrease inviscosity was observed visually. This indicates that the emulsion hadcollapsed from an oil/water emulsion to a water/oil emulsion. The totalamount of oil added before the "end point" was found by weighing theblender jar.

Emulsifying capacity was calculated as g oil per gram protein (N×6.38).

The emulsifying capacity was measured five times for each product tocalculate an average capacity.

The result of this test is presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        Emulsifying capacity                                                          ______________________________________                                        Raw material  231 g oil/g protein                                             DH = 2%       467 g oil/g protein                                             DH = 4%       455 g oil/g protein                                             DH = 6%       507 g oil/g protein                                             ______________________________________                                    

In the second method, the emulsifying properties were examined in amodel food system for a liquid mother milk substitute product of thefollowing composition:

    ______________________________________                                        Protein/protein hydrolysate                                                                         2.0%                                                    (appr. 80% protein dry substance)                                             Maltodextrin DE 8-10 (CPC-1908)                                                                     3.5%                                                    Sucrose               3.5%                                                    Oil (soy)             3.5%                                                    Water                 87.5%                                                   ______________________________________                                    

The protein/protein hydrolysate, maltodextrin and sucrose were dissolvedin water and heated to approximately 70° C. The oil was heated toapproximately 70° C. Using a Silverson-mixer the water-phase was mixed,and during mixing oil was added to obtain a pre-emulsification by mixingfor 2 minutes.

The mixture was homogenised using a Rannie Homogenizer at approximately70° C. and 300 bar, before the emulsion was heat-treated at 85° C. for 3minutes and then cooled to below 20° C. Samples were stored cold, below5° C.

After 1 and 7 days of storage, the emulsions are judged visually. If thesamples are inhomogen, or free fat is on the surface, the emulsifyingcapacity is poor.

Moreover, after 1 and 7 days of storage, the emulsions are centrifugedat 15,000 xg for 15 minutes and judged visually. If samples areinhomogen, or free fat is on the surface, the emulsifying capacity ispoor.

In this experiment, no free fat on the surface of any of the samples wasobserved, indicating stable emulsions with an excellent emulsifyingcapacity.

EXAMPLE 4

preparation of high viscosity soy protein

4000 ml of a suspension of soy protein isolate (Purina 500E, ProteinTechnology Int., USA), which suspension contained about 8% protein(N×6.25) were subjected to incubation with SP 446 protease (1% of the 27CPU/g enzyme), at a pH of 8.0 and a temperature of 65° C.

During incubation, which was monitored by means of a pH-stat(Radiometer, Copenhagen, Denmark), the pH was kept constant by additionof 4N NaOH. The reaction was monitored by base consumption, viscosityand osmolality.

The result of this preparation is presented on FIG. 6. A local viscositymaximum is noticed after 180 minutes of reaction, similar to incubationon whey protein. However, the viscosity at this point is far from thestarting value indicating a very limited plastein synthetic activity onsoy protein.

EXAMPLE 5

Molecular Weight Distribution

A sample of the incubated whey protein obtained according to Ex. 2 wasdiluted, filtrated and injected into a liquid chromatographic system,operating in the Gel Permeation Chromatography (GPC) mode.

This separation technique utilizes a liquid flow through a column filledwith porous particles, having pores with a well-defined pore diameter.When a solution of peptides having different molecular size passesthrough the column, the small peptides will be able to flow into thepores while the larger peptides will be excluded from the pores. Thus,the peptides in a solution will be separated according to molecular size(and weight), as the larger peptides will be eluted faster from thecolumn than the smaller peptides.

A detector at the column outlet continuously measures the effluent. Thechromatographic system is calibrated with peptides with known molecularweight.

Chromatographic equipment

The HPLC system consisted of a High Pressure pump, Waters M 510, at aflow rate of 0.7 ml/min. The injector was a Waters WISP M 710, and thedetector a Waters M 440 with wavelength extension to 214 nm.

Three GCP columns, TSK G 2000 SWXL, 7.8 mm ×300 mm, were connected inseries and operated at ambient temperature.

Integration/data processing were performed on a Waters 820MAXIMA SIMchromatography data system with 810/820 GPC option.

As mobile phase a mixture of 0.05M phosphate buffer and 0.5Mammoniumchloride solution containing 0.1% trifluoroacetic acid and 25%acetonitrile was used.

Calibration

The chromatographic system was calibrated by means of injections ofnumerous peptide standards with known molecular weight. The molecularweight of each standard is plotted semilogarithmically versus theobserved volume of phase needed to eluate the peptide from the column.By a least squares calculation, the best fitting 3'd order polynomiumwas calculated. This curve represents the calibration curve.

Analysis

The sample was diluted/dissolved in mobile phase to approximately 5mg/ml. The solution was filtered through 22 μm filter and 20 μl wereused for injection into the chromatograph. The detector response versuselution volume was recorded.

The recorded curve, the chromatogram, shows the actual molecular weightdistribution of the sample. To allow for calculations as to accumulatedweight distribution and average molecular weight calculations, thechromatogram was divided into small time (and elution volume) segments,each segment characterized by the elution volume and the area of thechromatogram over the time interval.

Calculation

The results appear from FIG. 7. The results are given in terms ofweight-and number-average molecular weights, calculated by the followingexpressions: ##EQU3## where: M_(w) =Weight average molecular weight, and

M_(n) =Number average molecular weight.

A_(i) =Area of chromatogram for each segment, measured as theaccumulated detector response over each time interval.

M_(w),i =The corresponding molecular weight for each segment. The valueis calculated by means of the calibration curve, using the averageelution volume over the time interval.

From these calculations the weight average molecular weight wascalculated as 2730, and the number average molecular weight wascalculated as 1220, indicating an average peptide length<×>of 10.3.

FIG. 8 presents the distribution of peptides by weight and by number.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 222 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerValIleGlySerAspAspArgThrArgValThrAsnThrThrAla                              151015                                                                        TyrProTyrArgAlaIleValHisIleSerSerSerIleGl ySerCys                             202530                                                                        ThrGlyTrpMetIleGlyProLysThrValAlaThrAlaGlyHisCys                              35404 5                                                                       IleTyrAspThrSerSerGlySerPheAlaGlyThrAlaThrValSer                              505560                                                                        ProGlyArgAsnGlyThrSerTyrProTyrGlySerValLysSerThr                              65707580                                                                      ArgTyrPheIleProSerGlyTrpArgSerGlyAsnThrAsnTyrAsp                              8590 95                                                                       TyrGlyAlaIleGluLeuSerGluProIleGlyAsnThrValGlyTyr                              100105110                                                                     PheGlyTyrSerTyrThrThrSerSerLeuValGlyT hrThrValThr                             115120125                                                                     IleSerGlyTyrProGlyAspLysThrAlaGlyThrGlnTrpGlnHis                              130135140                                                                     SerGlyProIleAlaIleSerGluThrTyrLysLeuGlnTyrAlaMet                              145150155160                                                                  AspThrTyrGlyGlyGlnSerGlySerProValPhe GluGlnSerSer                             165170175                                                                     SerArgThrAsnCysSerGlyProCysSerLeuAlaValHisThrAsn                              180185 190                                                                    GlyValTyrGlyGlySerSerTyrAsnArgGlyThrArgIleThrLys                              195200205                                                                     GluValPheAspAsnLeuThrAsnTrpLysAsn SerAlaGln                                   210215220                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       SerValIleGlySerAspAspArgThrArgValThrAsnThrThrAla                              151015                                                                        TyrMetThrArg                                                                  20                                                                        

We claim:
 1. A method for making plastein material comprising(a) adding an enzyme preparation substantially free of subtilisin A activity to the reaction mixture, said reaction mixture comprising a proteinaceous substrate at a concentration of 5-50% (w/w), said enzyme preparation comprising a proteolytic enzyme which:(i) is a serine protease specific for glutamic acid and aspartic acid residues; (ii) has a specific activity of at least 25 CPU per gram of enzyme protein and an apparent molecular weight of about 23,600; (III) is inhibited by diisopropyl phosphofluroridate, but not phenylmethane sulfonylfluoride; (iv) and exhibits at least 75% of its maximum activity in the pH range of 6.5-10.0; (v) has an N-terminal sequence of SEQ ID NO: 2 (b) incubating the reaction mixture of step (a) at a temperature of 20°-70° C., and at a pH of 4-12; and (c) inactivating said proteolytic enzyme in the reaction mixture of step (b).
 2. The method according to claim 1 in which the proteinaceous substrate is casein, soy protein or whey protein.
 3. The method according to claim 1 in which the proteinaceous substrate is whey protein or a proteinaceous mixture comprising whey protein.
 4. The method according to claim 1 in which the proteinaceous substrate is a proteinaceous mixture comprising whey protein and casein.
 5. The method according to claim 1 in which the proteinaceous substrate is a proteinaceous mixture comprising whey protein and soy protein.
 6. The method according to claim 1 in which the proteinaceous substrate is a proteinaceous mixture comprising casein and soy protein.
 7. The method according to claim 1 in which the proteinaceous substrate is present at a concentration of 5-30% (w/w).
 8. The method according to claim 1 in which the proteinaceous substrate is present at a concentration of 5-15% (w/w).
 9. The method according to claim 1 in which the proteolytic enzyme is added to the reaction mixture at a concentration of 0.05-50 CPU/100 g protein.
 10. The method according to claim 1 in which the proteolytic enzyme is added to the reaction mixture at a concentration of 0.1-25 CPU/100 g protein.
 11. The method according to claim 1 in which the proteolytic enzyme is added to the reaction mixture at a concentration of 1-25 CPU/100 g protein.
 12. The method according to claim 1 in which the reaction mixture in step (b) is incubated at a temperature of 45°-65° C.
 13. The method according to claim 1 in which the reaction mixture in step (b) is incubated at a pH of 6-9.
 14. The method according to claim 1 in which the reaction mixture in step (b) is incubated for 0.5-24 hours.
 15. The method according to claim 1 in which the reaction mixture in step (b) is incubated for 1-10 hours.
 16. The method according to claim 1 in which the reaction mixture in step (b) is incubated for 1-5 hours.
 17. The method according to claim 1 which further comprises adding at least one amino acid to the proteinaceous substrate prior to step (b). 